Major technological breakthroughs in biology reveal new dimensions in our understanding of gene regulation and chromatin 3-D structure. At the heart of understanding gene regulation are studies of transcription-regulating, long non-coding RNAs (lncRNAs) that are transcribed from enhancers. While the concept that lncRNAs play genome-wide roles as transcriptional regulators is now accepted, the relationship between lncRNAs and chromatin 3-D structure is still unclear. Therefore, a central question in biology remains: How do lncRNAs interact with chromatin to establish gene regulatory networks in the context of chromatin 3-D structure? By using a combination of large-scale analyses, including next-generation nucleic-acid sequencing, mass spectrometry protein sequencing, genome-wide chromosome conformation, and bioinformatics, we can now investigate the relationship between epigenetic factors, gene regulation, and chromatin 3-D structure. For over a decade, my lab has been studying transcriptional regulation by Evf2, the first enhancer-regulating, ultraconserved lncRNA (Feng et al. 2006, Bond et al. 2009, Berghoff et al. 2013, Cajigas et al. 2015). Collectively, these studies establish some fundamental principles regarding the role of lncRNAs in gene regulation, including the finding that a single developmentally regulated lncRNA controls GABAergic interneuron development and adult brain circuitry (Bond et al. 2009). Our previous work focused on Evf2 regulation of adjacent or overlapping, Dlx5/6 intergenic enhancers, and reported short-range effects on gene expression, transcription factor recruitment, histone modifications, site-specific CpG DNA methylation, and direct inhibition of ATPase and chromatin remodeling activities (Bond et al. 2009, Berghoff et al. 2013, Cajigas et al. 2015). In this proposal, we will combine the genetic and biochemical tools developed in my lab over the past decade, with recent large-scale technologies, to determine the relationship between the Evf2 gene regulatory network and chromatin 3-D structure.